Project Summary Vascular migration is a hallmark vascular pathology underlying atherosclerosis and restenosis following vascular injury, which are the major causes of mortality in Veteran population. Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis. Little is known regarding mechanisms involved in this response. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1, but also by the copper exporter ATP7A whose function is mediated through copper-dependent translocation from trans-Golgi network (TGN) as well as copper chaperon, antioxidant-1 (Atox1) which obtains copper from CTR1 and transfer it to ATP7A. Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration and neointimal formation. Most recently, our laboratory demonstrated that PDGF stimulates copper- and CTR1-dependent translocation of ATP7A from TGN to the lipid rafts localized at the leading edge where it recruits Rac1 as well as decreases copper content and secretory copper enzyme, pro-lysyl oxidase (pro-LOX). This in turn stimulates lamellipodia formation and LOX activity, thereby promoting VSMC migration. Underlying molecular events remain unknown. Our preliminary studies identified a scaffold protein IQGAP1 as a novel binding partner for ATP7A. IQGAP1 is shown to bind directly to active form of Rac1 to keep it active state and involved in cell motility. Our preliminary data are consistent with the novel hypothesis that CTR1-Atox1 pathway and ATP7A binding to IQGAP1 plays an important role PDGF-induced copper-dependent ATP7A and Rac1 translocation to the leading edge, lamellipodia formation and VSMC migration. Moreover, caveolae/lipid rafts are important signaling domains where PDGF stimulates ATP7A- dependent LOX activation, which is involved in copper-dependent VSMC migration. To test this, three specific aims will be proposed. Aim 1 will determine the molecular mechanism by which PDGF stimulates ATP7A and Rac1 translocation to the leading edge, which is involved in lamellipodia formation and VSMC migration. We will identify ATP7A-IQGAP1 binding sites using in vitro pull-down or in vivo co-transfection assays and define the functional significance of their binding in PDGF-induced responses. FRET analysis will be used to examine role of copper transporters in regulating Rac1 activity and translocation in live cell image analysis. Aim 2 will determine the functional significance of ATP7A movement to caveolin-enriched lipid rafts in PDGF-induced secretion of pro-LOX and copper homeostasis, which are required for LOX activation and VSMC migration. Subcellular fractionation, VSMC derived from caveolin-1 deficient mice, 64Cu metabolic labeling analysis, inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence microscope will be used. Aim 3 will assess the functional role of ATP7A and its regulators in neointimal formation in response to vascular injury. ATP7A mutant mice, and IQGAP1 deficient mice and wire injury model will be used. These studies will provide new insight into copper transporters as potential therapeutic targets for cardiovascular diseases such as atherosclerosis.